The Biosynthetic Pathways for Shikimate and Aromatic Amino ... · a single AtDHQ/SDH gene, tobacco plants possess two genes. RNAi-mediated suppression of either of the two tobacco

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The Biosynthetic Pathways for Shikimate and Aromatic Amino Acids inArabidopsis thalianaAuthors: Vered Tzin, and Gad GaliliSource: The Arabidopsis Book, 2010(8) Published By: American Society of Plant BiologistsURL: https://doi.org/10.1199/tab.0132

Downloaded From: https://bioone.org/journals/The-Arabidopsis-Book on 14 Apr 2019Terms of Use: https://bioone.org/terms-of-use

The Arabidopsis Book © 2010 American Society of Plant Biologists

First published on May 17, 2010 10.1199/tab.0132

INTRODUCTION

The aromatic amino acids (AAA), phenylalanine (Phe), tyrosine (Tyr) and tryptophan (Trp) (Fig. 1), are central molecules in plant metabolism. Besides their function as building blocks of proteins, the three AAA serve as precursors for a variety of plant hormones, such as auxin and salicylate, as well as for a very wide range of aromatic secondary metabolites with multiple biological functions and biotechnological value in the health promoting, medical and food industries (Bartel, 1997; Vogt, 2010). The AAA of plants are also essential nutritive compounds in the diets of humans and monogastric livestock, which are unable to synthesize them (Li and Last, 1996; Galili et al., 2002). Additionally, the shikimate pathway enzyme 5-enolpyruvylshikimate-3-phospate synthase (EPSP synthase) is the target of the glyphosate herbicide, and non-plant EPSP synthase provides the herbicide-resistance trait in a number of commercial transgenic crops (Duke and Powles, 2008). These important properties account for the major motiva-tion to elucidate the regulation of the shikimate and AAA biosyn-thesis pathways in plants.

The biosynthesis of AAA from core primary metabolism initi-ates via the shikimate pathway, leading to the synthesis of cho-rismate (Fig. 2). Chorismate is the initial branch point metabolite in the synthesis of all three AAA (Fig. 2) and the wide range of aromatic secondary metabolites derived from it (Gilchrist and Kosuge, 1980; Herrmann, 1995). Hence, the shikimate and AAA

The Biosynthetic Pathways for Shikimate and AromaticAmino Acids in Arabidopsis thaliana

Vered Tzina and Gad Galili a,1

aDepartment of Plant Sciences, The Weizmann Institute of Science, Rehovot 76100 Israel.1Address correspondence to [email protected]

The aromatic amino acids phenylalanine, tyrosine and tryptophan in plants are not only essential components of pro-tein synthesis, but also serve as precursors for a wide range of secondary metabolites that are important for plant growth as well as for human nutrition and health. The aromatic amino acids are synthesized via the shikimate pathway followed by the branched aromatic amino acid metabolic pathway, with chorismate serving as a major branch point intermediate metabolite. Yet, the regulation of their synthesis is still far from being understood. So far, only three en-zymes in this pathway, namely, chorismate mutase of phenylalanine and tyrosine synthesis, tryptophan synthase of tryptophan biosynthesis and arogenate dehydratase of phenylalanine biosynthesis, proved experimentally to be allo-sterically regulated. The major biosynthesis route of phenylalanine in plants occurs via arogenate. Yet, recent studies suggest that an alternative route of phynylalanine biosynthesis via phenylpyruvate may also exist in plants, similarly to many microorganisms. Several transcription factors regulating the expression of genes encoding enzymes of both the shikimate pathway and aromatic amino acid metabolism have also been recently identifi ed in Arabidopsis and other plant species.

Figure 1. Structures of chorismate and the three aromatic amino acids.

biosynthesis pathways also represent a major regulatory link of primary and secondary metabolism in plants.

Despite the extreme signifi cance of the AAA to the life cycles of plants, the regulation their biosynthesis via the shikimate and AAA biosynthesis pathways has been largely ignored and even not reviewed in the last decade. Yet, these biosynthesis pathways have been re-visited in recent years by a number of studies. The present review focuses on new insights into the regulation of AAA biosynthesis, which are based on: (i) recent studies, focusing mainly on Phe and to a smaller extent also on Tyr and Trp biosyn-

: e0132.


2 of 18 The Arabidopsis Book

thesis; and (ii) gene sequence data generated from the sequenc-ing of the entire Arabidopsis thaliana (Arabidopsis) genome. A more extensive background on the biochemistry of the shikimate and AAA biosynthesis pathways is available in the following out-standing and most recent reviews dating to the years 1995 and 1999 (Herrmann, 1995; Herrmann and Weaver, 1999).

THE SHIKIMATE PATHWAY

The shikimate pathway, also known as the chorismate biosyn-thesis pathway, converts two metabolites, phosphoenolpyruvate (PEP) of the glycolysis pathway and erythrose 4-phosphate (E4-P) of the non-oxidative branch of the pentose phosphate pathway, into chorismate (Fig. 2). Genes encoding enzymes of the entire shikimate pathway have been identifi ed in Arabidopsis and other plant species, mostly due to their homology to shikimate path-way genes from microbial organisms. The conversion of PEP and E4-P to chorismate comprises seven reactions catalyzed by six enzymes. The fi rst enzyme of the shikimate pathway is 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAHPS) (EC 2.5.1.54) converting PEP and E4-P into 3- dehydroquaianate (Fig. 2). Arabidopsis plants possess two known DAHPS genes:

AtDAHPS1 (At4g39980) and AtDAHPS2 (At4g33510) in addi-tion to one putative gene (At1g22410) with high similarity to At-DAHPS1. Expression of AtDAHPS1 in Escherichia coli showed that this enzyme requires Mn2+ and reduced thioredoxin (TRX) for activity, thereby, linking carbon fl ow into the shikimate pathway to electron fl ow from photosystem I (Entus et al., 2002). Desp ite the metabolic importance of DAHPS as a branch point metabolite converting primary carbon metabolism into the shikimate path-way, it is still unknown whether this enzyme serves as a major regulator of fl ux between primary and secondary metabolism in plants. DAHPS activity may however be central to the ability of the shikimate pathway to compete for PEP and E4-P with glycoly-sis as well as with the non-oxidative pentose phosphate pathway (Fig. 2).

The second enzyme of the shikimate pathway is 3-dehydro-quinate synthase (DHQS; EC 4.2.3.4; At5g66120), which con-verts 3-deoxy-d-arabino-heptulosonate-7-phosphate into 3-de-hydroquinate (Fig. 2). The third and fourth enzymatic steps are catalyzed by the bi-functional enzyme 3-dehydroquinate dehy-dratase/shikimate 5-dehydrogenase (DHQ/SDH; EC 4.2.1.10 and EC 1.1.1.25) (At3g06350), leading to the formation of shikimate (Fig. 2). This bifunctional enzyme has been characterized in to-mato (Solanum lycopersicum) (Bischoff et al., 2001) and tobacco (Nicotiana tabacum) (Bonner and Jensen, 1994). A recent study showed that the Arabidopsis AtDHQ/SDH gene is required for female gametophyte development and function (Pagnussat et al., 2005). The crystal structure of Arabidopsis DHQ/SDH with shikimate bound at the SDH site and tartrate at the DHQ site has recently been elucidated (Singh and Christendat, 2006). The interactions observed in the DHQ-tartrate complex reveal a con-served mode for substrate binding between the plant and micro-bial DHQ dehydratase family of enzymes. The arrangement of the two functional domains of this enzyme suggests that the control of metabolic fl ux through the shikimate pathway is achieved by in-creasing the effective concentration of the intermediate substrate, 3-dehydroshikimate, through the proximity of the two sites (Singh and Christendat, 2006). While Arabidopsis plants possess only a single AtDHQ/SDH gene, tobacco plants possess two genes. RNAi-mediated suppression of either of the two tobacco DHQ/SDH-1 and NtDHQ/SDH-2 genes caused differential steady state levels of the pathway substrates dehydroquinate and shikimate (Ding et al., 2007).

The fi fth enzymatic step of the shikimate pathway is catalyzed by shikimate kinase (SK) (EC 2.7.1.71), which converts shikimate to shikimate 3-phosphate (Fig. 1). Arabidopsis plants possess two SK isoforms: AtSK1 (At2g21940) and AtSK2 (At4g39540) as well as two additional SK-like genes that arose from an ancestral plant SK gene duplicates, but lost their SK activity (Fucile et al., 2008). It has been suggested that these two genes may have evolved a new enzymatic function that is not related to the shikimate path-way (Fucile et al., 2008). Several lines of evidence suggest that plant SK acts as a regulatory step for the shikimate pathway, fa-cilitating metabolic fl ux towards specifi c pools of secondary me-tabolite. These include: (i) a rapid induction of plant SK transcripts by fungal elicitors (Gorlach et al., 1995); (ii) a signifi cant sensitiv-ity of plant SK activity to cellular ATP energy charge; and (iii) the differential expression of the three rice (Oryza sativa) SK genes in specifi c developmental stages and in response to biotic stress (Kasai et al., 2005).

Figure 2. The shikimate pathway. Enzymes involved in the biosynthesis of chorismate.


Aromatic Amino Acid Biosynthesis in Arabidopsis 3 of 18

The sixth enzymatic step of the shikimate pathway is cata-lyzed by 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) (CE 2.5.1.19), which leads to the synthesis of enolpyruvylshiki-mate 3-phosphate (EPSP) (Fig. 1). The Arabidopsis EPSPS is en-coded by one functional gene (At2g45300) and perhaps also by a second putative gene (At1g48860) (Klee et al., 1987). This en-zyme has been broadly studied for the last ~30 years (for review see Duke and Powles, 2008) due to its association with resis-tance to the herbicide N-phosphonomethylglycine (glyphosphate, an analog of phosphoenylpyruvate), which is the basis for the Roundup-Ready transgenic crops (Singer and McDaniel, 1985; Smart et al., 1985; Stalker et al., 1985). The native plant EPSPS is competitively inhibited by the herbicide glyphosphate, the con-sequence of which is a diminished fl ux of the shikimate pathway (Healy-Fried et al., 2007).

The fi nal step in the shikimate pathway is catalyzed by choris-mate synthase (CS) (CE 4.2.3.5), which converts EPSP to cho-rismate (Fig. 2). This enzyme was fi rst characterized in Coryda-lis semoervirens (Schaller et al., 1991) and is proposed to have been derived from a common ancestor for bacteria, plants and fungi (Macheroux et al., 1999). Arabidopsis possesses a single CS gene (At1g48850), in contrast to tomato plants, which pos-sess two differentially expressed CS genes, termed LeCS1 and LeCS2 (Gorlach et al., 1993).

CHORISMATE, A CENTRAL BRANCH POINT METABOLITE IN THE SYNTHESIS OF AROMATIC AMINO ACIDS ANDSECONDARY METABOLITES

Chorismate, the terminal metabolite of the shikimate pathway serves as the initiator metabolite for the synthesis of the three AAA (Fig. 3) and hence also for the various aromatic secondary metabolites derived from them. Yet, chorismate also serves one of the initiator substrate of the synthesis of a number of other aromatic metabolites, many of which are likely to be still unknown.

Some examples of chorismate-derived metabolites are: (i) cho-rismate is one of the precursor metabolites for the synthesis of tetrahydrofolate (vitamin B9; also commonly termed folate), serv-ing as the substrate of the aminodeoxychorismate synthase (Fig. 3) (Basset et al., 2004; Waller et al., 2010); (ii) chorismate is con-verted to isochorismate by isochorismate synthase (Wildermuth et al., 2001) on route to the production of salicylate (SA) (Fig. 3) (Garcion et al., 2008); and (iii) chorismate also serves the precur-sor metabolite for the synthesis of phylloquinone (vitamin K1) and many other plant pigments (Gross et al., 2006; Kim et al., 2008). Hence, chorismate is one of the central branch point metabolites in plant cells.

THE BIOSYNTHESIS NETWORK OF THE THREE AROMATIC AMINO ACIDS PHE, TYR AND TRP

The unsolved pathway of Phe biosynthesis: two possible metabolic routes using arogenate or phenylpyruvate as intermediates

The fi rst committed step of Phe biosynthesis from chorismate is catalyzed by chorismate mutase (CM) (CE 5.4.99.5), which con-verts chorismate to prephenate (Fig. 4). Three CM genes have so far been described in Arabidopsis, namely AtCM1 (At3g29200), AtCM2 (At5g10870) and AtCM3 (At1g69370) (Mobley et al., 1999). The three genes are differentially expressed in various tis-sues and the expression of only AtCM1 is induced by various elicitors and pathogens (Mobley et al., 1999; Ehlting et al., 2005). The activities of the three Arabidopsis CM isoforms were demon-strated by complementing E. coli and yeast CM-defi cient strains (Eberhard et al., 1993; Eberhard et al., 1996). The activities of AtCM1 and AtCM3 are inhibited by Phe and Tyr, whereas the ac-tivity of AtCM2 appears to be insensitive to these amino acids (Eberhard et al., 1996). The fi nal two enzymatic steps converting prephenate to Phe in plants are still not entirely elucidated. The major route involves the conversion of chorismate via arogenate to Phe, catalyzed by respective enzymes prephenate aminotrans-ferase (PAT) (CE 2.6.1.79) and arogenate dehydratase (ADT) (CE 4.2.1.49) (Cho et al., 2007; Yamada et al., 2008; Maeda et al., 2010)(Fig. 4). Yet, it is still not clear whether plants can also con-vert chorismate to Phe via phenylpyruvate (PPY), using enzymes with prephenate dehydratase (PDT) and Phe aminotransferase activities (Fig. 4) in a similar manner to E. coli and various other microorganisms. A PAT enzymatic activity, converting prephenate into arogenate (Fig. 4), has been reported in plants (Siehl et al., 1986; De-Eknamkul and Ellis, 1988). Yet, no plant gene encoding such an activity has so far been reported. An in silico data mining approach identifi ed six putative ADT genes in Arabidopsis, name-ly, ADT1 (At1g11790), ADT2 (At3g07630), ADT3 (At2g27820), ADT4 (At3g44720), ADT5 (At5g22630) and ADT6 (At1g08250). Biochemical characterization of the recombinant enzymes en-coded by these six Arabidopsis genes suggested that all of them possess arogenate dehydratase activity, converting arogenate into Phe (Fig. 4). Yet, three of them (ADT1, ADT2 and ADT6) can also utilize prephenate as a substrate and convert it to PPY (Fig. 4), even though they exhibit a preference for arogenate (Cho et al., 2007). A rice 5-methyl-Trp resistant mutant, called Mtr1, which over-accumulates Phe, Trp and several phenylpropanoids,

Figure 3. Chorismate, a central branch point metabolite in the synthesis of aromatic amino acids and secondary metabolites. First enzymes involved in several secondary pathways derived from chorismate.



appeared to result from a point mutation in a gene encoding an enzyme possessing both ADT and PDT activities, rending these activities insensitive to feedback inhibition by Phe (Yamada et al., 2008). Nevertheless, similar to the Arabidopsis enzymes that can utilize both ADT and PDT substrates, this rice enzyme possessed a preference to arogenate, implying that it functions primarily as an ADT. Recently, three genes encoding ADT enzymes were identifi ed in petunia (Petunia hybrida). Similar to the Arabidop-sis ADT isozymes, the three petunia ADT isozymes preferentially use arogenate as a substrate, but can also use prephenate as a substrate at a much lower effi ciencies, supporting the hypothesis of preferential utilization of the arogenate route rather than the PPY route for Phe biosynthesis in plants (Maeda et al., 2010). However, feeding shikimate into petunia petals with suppressed expression of ADT1 (the major ADT enzyme in petunia) led to the accumulation of prephenate and PPY and also to partial recovery of the reduced Phe level, strongly indicating that petunia plants can also synthesize Phe via the PPY route.

To study the consequence of producing PPY in plants by metabolic engineering, we have recently expressed a bacterial PheA gene encoding a bi-functional CM/PDT enzyme that con-verts chorismate via prephenate to PPY (Tzin et al., 2009). These Arabidopsis plants had a signifi cant increase in the level of Phe, with no increase in the level of PPY. Although it is likely that a con-siderable amount of the prephenate, produced by the CM activity of the bacterial CM/PDT enzyme, was converted via arogenate to Phe using the ADT enzyme (Fig. 4), the fact that these plants showed no increased level of PPY suggests that Arabidopsis ap-parently possesses an endogenous AAAT activity that can use PPY as a substrate and covert it to Phe (Tzin et al., 2009) (Fig. 4). Yet, no gene encoding an aromatic amino acid aminotransferase (AAAAT) (CE 2.6.1.57) that can specifi cally convert PPY into Phe has so far been identifi ed in plants. Hence, taken together, the studies described above imply that plants use primarily the aroge-nate route for the synthesis of Phe, although some minor function of the PPY route in Phe biosynthesis cannot be ruled out. This is also supported by the observation that a number of plants spe-

cies contain PPY, which also serves as a precursor for a number of secondary metabolites such as phenylacetaldehyde, 2-phenyl-ethanol and 2-phenylethyl b-d-glucopyranoside (Watanabe et al., 2002; Kaminaga et al., 2006).

The pathway of Tyr biosynthesis

The major route of Tyr biosynthesis initiates from chorismate, using the same fi rst two enzymes of Phe biosynthesis, namely CM and PAT, to produce arogenate (Fig. 4 and 5). Arogenate is then converted into Tyr by arogenate dehydrogenase (TyrA) (CE 1.3.1.43) (Fig. 5). TyrA activity has been demonstrated in tobacco(Gaines et al., 1982), maize (Byng et al., 1981), sorghum (Con-nelly and Conn, 1986) and Arabidopsis (Rippert and Matringe, 2002b). In Arabidopsis plants, two genes encoding TyrA enzymes were identifi ed: TyrA1 (At5g34930) and TyrA2 (At1g15710) (Rip-pert and Matringe, 2002b, a; Rippert et al., 2009).

A second possible route of Tyr biosynthesis has also been suggested, which includes the conversion of prephenate to p-hydroxyphenylpyruvate (p-hydroxyPPY) by prephenate dehydro-genase (PDH) (CE 1.3.1.43), which may be catalyzed by TyrA2 (Rippert and Matringe, 2002b). Subsequently, p-hydroxyPPY converts to Tyr by a broad range AAAAT (Fig. 5). Nevertheless, at a non-saturating concentration of prephenate, TyrA2 enzyme activity is 2000 times less effi cient in catalyzing the reaction with prephenate than with arogenate (Rippert and Matringe, 2002a), and therefore the possible existence of this alternative route for Tyr biosynthesis using PDH is still in doubt.

The pathway of Trp biosynthesis

The fi rst committed step of Trp biosynthesis includes a transfer of an amino group of glutamine to chorismate to generate anthrani-late and pyruvate, catalyzed by anthranilate synthase (AS) (CE 4.1.3.27) (Fig. 6). Purifi ed plant AS holoenzymes are believed to

Figure 4. The pathway of Phe biosynthesis. Enzymes involved in the biosynthesis of Phe. N.D. not detected in Arabidopsis plants.



be heterotetramers composed of two alpha and two beta sub-units (Niyogi et al., 1993; Poulsen et al., 1993). The Arabidopsis genome possesses two functional genes encoding the AS alpha subunit, ASa1 (At5g05730) and ASa2 (At2g29290), as well as a single functional ASb1 gene (At1g25220) encoding the AS beta subunit. In addition, two other genes were putatively assigned as encoding ASa subunits (At2g28880 and At3g55870) and ad-ditional fi ve genes were putatively assigned as encoding ASb subunits (At5g57890, At1g25155, At1g24807, At1g24909 and At1g25083). Interestingly, four of the putative genes encoding ASb are located on one cluster on chromosome 1 (for more de-tails see http://www.plantcyc.org). The alpha subunit possesses the catalytic activity and the beta subunit possesses an amino-transferase activity, which transfers an amino group from gluta-mine to the alpha subunit. AS activity in plants is feedback inhib-ited by Trp through binding of Trp to the alpha subunit. Expression of AS genes encoding feedback-insensitive enzymes in a variety of plant species generally increases the production of free Trp and secondary metabolites derived from it (Li and Last, 1996; Tozawa et al., 2001; Hughes et al., 2004). The trp4 mutation in the gene encoding the Arabidopsis ASb1 subunit suppresses ac-cumulation of the product of this enzyme, anthranilate (Niyogi et al., 1993). Anthranilate possesses a strong blue fl uorescence un-der UV light, which has been utilized as a phenotypic marker for indentifying Arabidopsis mutants in the Trp biosynthesis enzymes (Rose et al., 1992; Radwanski et al., 1995).

The second enzyme in the Trp biosynthesis pathway is an-thranilate phosphoribosylanthranilate transferase (PAT1) (CE 2.4.2.18; At5g17990), which converts anthranilate and phospho-ribosylpyrophosphate into phosphoribosylanthranilate and inor-ganic pyrophosphate (Fig. 6).

The third enzyme in the Trp biosynthesis pathway is phospho-ribosylanthranilate isomerase (PAI) (CE 5.3.1.24), which converts phosphoribosylanthranilate into l-(O-carboxyphenylamino)-l-de-oxyribulose-5-phosphate (CDRP) (Fig. 6). Arabidopsis possess-es three genes encoding PAI isoforms; PAI1 (At1g07780), PAI2 (At5g05590) and PAI3 (At1g29410).

Figure 5. The pathway of Tyr biosynthesis. Enzymes involved in the biosynthesis of Tyr. N.D. not detected in Arabidopsis plants.

Figure 6. The pathway of Trp biosynthesis. Enzymes involved in the bio-synthesis of Trp.



The fourth enzyme of Trp biosynthesis is indole-3-glycerol phosphate synthase (IGPS) (EC 4.1.1.48), which catalyzes the conversion of 1-(O- carboxyphenylamino)-1-deoxyribulose-5-phosphate to indole-3-glycerol phosphate (Li et al., 1995a). Ara-bidopsis plants possess one gene encoding a functional IGPS (AT2G04400) and also a second gene (AT5G48220) encoding a putative IGPS (Li et al., 1995a). IGPS is an important enzyme in the biosynthesis of Trp and the hormone indole-3-acetic acid (IAA; auxin) because it is the only known enzyme that catalyzes the for-mation of the indole ring. Quantitative comparison of the relative levels of Trp and IAA content in different Arabidopsis Trp biosyn-thesis mutants as well as in transgenic plants expression an IGPS antisense construct indicates that indole-3-glycerol phosphate is the branch-point metabolite for a de novo Trp-independent IAA biosynthesis in Arabidopsis (Ouyang et al., 2000). Interestingly, in both fungi and bacteria, IGPS is synthesized as a fusion protein containing one or two other enzymes of the Trp biosynthesis path-way (Li et al., 1995b). However, in plants IGPS generally appears as a mono-functional enzyme based on its cDNA sequence and functional complementation analysis (Li et al., 1995a).

The last two steps in the Trp biosynthesis are catalyzed by Trp synthase (TS) (CE 4.2.1.20), which includes both alpha (TSa) and beta (TSb) subunits. Indole-3-glycerol phosphate is cleaved by TSa to indole and glyceraldehyde-3-phosphate (α-reaction). Then, indole is transported to TSb, which catalyzes its condensa-tion with serine (β-reaction) to produce Trp (Miles, 2001; Weber-Ban et al., 2001). Arabidopsis possesses at least one functional gene encoding TSa (At3g54640). Yet, a gene encoding a puta-tive TSa homolog (At4g02610), also named indole synthase, was identifi ed and characterized in Arabidopsis. Indole synthase possesses ~65% amino acid sequence identity to TSa (Zhang et al., 2008). Arabidopsis possess two genes encoding functional TSb subunits, namely, TSb1 (At5g54810) and TSb2 (At4g27070), as well as two additional genes encoding putative TSb subunits (At5g28237 and At5g38530). The function of TSa1 and TSb1 was demonstrated by the facultative Trp auxotroph mutants, trp3 and trp2, respectively (Last et al., 1991), and it was suggested that the TSa1 and TSb1 subunits form an active heterodimer (Radwanski et al., 1995). The Arabidopsis gene encoding TSa1 was cloned by functional complementation of an E. coli mutant and suggested to function as a monomer (Bohlmann et al., 1995; Radwanski and Last, 1995; Radwanski et al., 1995). Yet, whether TS activity oper-ates as a monomer or as a multi-enzyme complex is still not clear (Kriechbaumer et al., 2008).

TRANSCRIPTIONAL AND POST TRANSCRIPTIONALREGULATON OF THE SHIKIMATE PATHWAY AND AROMATIC AMINO ACID METABOLISM

Transcriptional regulation

Transcriptional regulation of the shikimate pathway and aromatic amino acid metabolism in plants has so far not been studied ex-tensively. The expression of DAHPS encoding the fi rst enzyme of the shikimate pathway (Fig. 2) is induced by physical wound-ing and methyl-jasmonate (Devoto et al., 2005; Yan et al., 2007), infi ltration with pathogenic Pseudomonas syringae strains (Keith et al., 1991), redox state (Entus et al., 2002) and abscisic acid

(Leonhardt et al., 2004; Catala et al., 2007). The expression of the gene encoding EPSPS is induced in response to infection by the necrotrophic fungal pathogen Botrytis cinerea (Ferrari et al., 2007) and by sulfate starvation (Nikiforova et al., 2003). Fun-gal elicitors also rapidly stimulate the production of mRNA of SK (Gorlach et al., 1995). Ozone treatment induces a signifi cant part of the shikimate pathway genes in tomato (Bischoff et al., 1996; Bischoff et al., 2001), tobacco (Janzik et al., 2005) and in the European beech (Fagus sylvatica) (Betz et al., 2009). Oligoga-lacturonides that are released from plant cell walls upon infec-tion with of the Botrytis cinerea pathogen stimulate a number of genes encoding enzymes of the shikimate and AAA biosynthe-sis pathways, as well as genes encoding enzyme of secondary metabolites derived from the AAA (Ferrari et al., 2007). The ex-pression of the three Arabidopsis genes encoding the three PAI isoforms (Fig. 6) is differentially regulated under normal growth conditions, with PAI1 and PAI3 showing ~10-fold higher expres-sion level than PAI2 (He and Li, 2001). Expression of these three PAI genes also respond differentially to environmental stresses, such as UV irradiation and treatment with the abiotic elicitor silver nitrate in a tissue- and cell-type-specifi c manner (Li et al., 1995b; He and Li, 2001). Deletion of the Arabidopsis gene encoding PAI1 causes some abnormal growth (He and Li, 2001) which indicates its predominant importance in Trp biosynthesis. Interestingly, the Arabidopsis PAI gene family is regulated by methylation in the Wassilewskija, but not Columbia ecotypes (Bender and Fink, 1995; Melquist et al., 1999). The PAI genes of Wassilewskija con-tain inverted repeats, which provide a trigger for their methylation (Bender and Fink, 1995; Melquist et al., 1999; Bartee and Bender, 2001; Melquist and Bender, 2003).

The Arabidopsis gene encoding IGPS of Trp biosynthesis (Fig. 6) is regulated by the hormones jasmonate (Sasaki-Sekimoto et al., 2005; Dombrecht et al., 2007) and salicylate (Rajjou et al., 2006), and also in seeds and seedlings by various defense mech-anisms (Job et al., 2005; Chibani et al., 2006). In addition, expres-sion of the Arabidopsis gene encoding PAT1 is apparently con-trolled by regulatory elements located inside introns, as inclusion of introns was shown to enhance the expression of PAT1-GUS fusion constructs that were stably transformed into Arabidopsis (Rose and Beliakoff, 2000).

Recently, in the frame of the AtGenExpress project, the re-sponse of the global Arabidopsis transcriptome to a variety of abiotic and biotic stresses was studied in roots and shoots, us-ing the Affymetrix ATH1 microarray (NASC; http://affymetrix.arabidopsis.info/) (Kilian et al., 2007). The database of these ex-periments was used in a bioinformatics study to analyze of the response of genes encoding biosynthesis enzymes as well as enzymes responsible for the fi rst catabolic enzymes of the dif-ferent amino acid in a variety of amino acid metabolic pathways (Less and Galili, 2008). The results showed that genes encod-ing amino acid catabolic enzymes principally respond in shorter time periods and are much more sensitive to abiotic stresses than genes encoding biosynthetic (allosteric and non-allosteric) enzymes. These responses also operated in a pathway-specifi c manner in response to different stress conditions (Less and Galili, 2008). These results imply that the catabolic genes play major regulatory roles in amino acid metabolism upon exposure to these stresses (Less and Galili, 2008). Interestingly, the Trp and Phe/Tyr branches of the AAA biosynthesis pathway responded



differently to UV-B stress. In the Trp biosynthesis pathway, UV-B stress stimulated the expression of the genes encoding both the biosynthesis enzymes and the catabolic enzymes CYP79B2 and CYP79B3. In contrast, this stress did not affect the expression of the genes encoding the biosynthesis enzymes of the Phe/Tyr branch, while it stimulated the expression of only the gene encod-ing the Tyr catabolism enzyme Tyr-aminotransferase, but not the Phe catabolism enzyme Phe-ammonia lyase (PAL). Interestingly, exposure of Arabidopsis plants to various stresses, including amino acid starvation, as well as to treatments with the oxidative stress-inducing herbicide acifl uorfen and the abiotic elicitor alpha-amino butyric acid, also induce the expression of genes encoding Trp biosynthesis enzymes (Zhao et al., 1998). Overexpression of members of two clades of Arabidopsis genes, encoding “altered Trp regulation1” [ATR1]-like and MYB28-like transcription factors in transgenic Arabidopsis stimulates the expression of specifi c genes belonging to both the shikimate and Trp biosynthesis path-ways, as well as genes encoding enzymes of Trp-derived sec-ondary metabolites (Malitsky et al., 2008). Similar results were also obtained upon expression of the petunia ODORANT1 gene, encoding a R2R3-type MYB transcription factor in petunia fl ow-ers (Colquhoun et al., 2010). Down-regulation of ODORANT1 in transgenic petunia plants strongly reduced the abundance of transcripts and metabolites from the shikimate pathway (Verdonk

et al., 2003). A functional homolog of ODORANT1 was not yet been identifi ed in Arabidopsis.

Post-translational regulation by enzyme feedback-inhibition loops

The activities of DAHPS enzymes (the fi rst enzymatic step of the shikimate pathway; see Fig. 2) from various microorganisms are generally regulated by allosteric feedback inhibition by the differ-ent AAA (Byng et al., 1983; Knaggs, 2001). In contrast, there is no published evidence showing that plant DAHPS enzymes are strongly allosterically inhibited in vivo by any of the AAA, and it is generally assumed that DAHPS activity in higher plants is not subject to a major allosteric control (Gilchrist and Kosuge, 1980; Herrmann and Weaver, 1999). Yet, the in vitro activities of DAHPSs from different plant species are weakly inhibited by Trp (Graziana and Boudet, 1980; Rubin and Jensen, 1985) and Tyr (Reinink and Borstap, 1982), or even can also be weakly acti-vated by either Trp or Tyr (Suzich et al., 1984; Pinto et al., 1986)(Fig.7). The activity of Vigna radiate (bean) DAHPS is weakly in-hibited by prephenate and arogenate, the precursor metabolites of Phe and Tyr biosynthesis (Fig. 7) (Rubin and Jensen, 1985), but whether this is due to inhibition of enzyme level or activity is

Figure 7. Post-transcriptional regulation of the shikimate pathway and aromatic amino acid metabolism. Key enzymes and metabolites are shown. Known allosteric regulation by compounds within the pathway is shown, activation with a green arrow, inhibition with a red line and bar, and putative allosteric inhi-bition with a dashed red line. DAHPS, 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase; ASa, anthranilate synthase alpha subunit; CM, chorismate mutase; PDT, prephenate dehydratase ; ADT, arogenate dehydratase; TyrA, arogenate dehydrogenase.



still unknown (Herrmann, 1995). It has also been suggested that the Petroselinum crispum (parsley) DAHPS activity results from several different isoforms, whose activities may be dependent on Mn2+ or Co2+ ions (McCue and Conn, 1989; Gorlach et al., 1993). In addition, the Mn2+-dependent regulation of DAHPS activity by arogenate was proposed as one of the key circuits in the overall pattern of allosteric control for the entire network of the shikimate and AAA biosynthesis (Doong et al., 1992; Doong et al., 1993). All in all, the above results imply that the shikimate pathway in plants is mostly regulated at the gene expression level rather than by post-translational controls.

The Regulation of AAA biosynthesis from chorismate by feed-back inhibition loops is primarily associated with: (i) the branch point enzymes AS and CM, which utilize the substrate choris-mate; (ii) the branch point enzyme ADT catalyzing the fi nal step in Phe biosynthesis; and (iii) the branch point enzyme TyrA catalyz-ing the fi nal step of Tyr biosynthesis (Fig. 7). AS, the fi rst enzyme specifi c for Trp biosynthesis, is feedback-inhibited by Trp (Fig. 7). Arabidopsis trp5 mutants, producing an ASa1 subunit that is in-sensitive to feedback inhibition by Trp, were isolated in the 1990s either by screening for accumulation of the intermediate metabo-lite anthranilate (through measuring its fl uorescent properties) or by resistance to toxic Trp analogs, such as 6-methyltryptophan (Kreps et al., 1996; Li and Last, 1996). CM, the fi rst specifi c en-zyme for Phe and Tyr biosynthesis, is normally feedback inhibited by Phe and Tyr and induced by Trp (Eberhard et al., 1996) (Fig. 7). To investigate the enzymatic properties of the three Arabidopsis CM isoforms, the Arabidopsis CM1, CM2 or CM3 cDNAs were expressed in yeast (Mobley et al., 1999). The activities of both the CM1 and CM3 isozymes were feedback inhibited by Phe and Tyr, while stimulated by Trp. In contrast, CM2 activity was insensitive to feedback inhibition by any of the AAA (Mobley et al., 1999). The activity of TyrA, the last enzyme of Tyr biosynthesis, is feedback inhibited by Tyr (Fig. 7) in Arabidopsis (Rippert and Matringe, 2002b) and Sorghum bicolor (Connelly and Conn, 1986). In ad-dition, ADT activity from tobacco, spinach, and S. bicolor was shown to be positively regulated by Tyr and negatively regulated by Phe (Jung et al., 1986; Siehl and Conn, 1988). However, the al-losteric regulation has not yet been characterized in Arabidopsis plants (Cho et al., 2007). In addition, the rice ADT is negatively regulated by Phe (Yamada et al., 2008), while its potential regula-tion by Tyr has not yet been elucidated.

Comparison of these feedback regulation loops shows that the fl ux from chorismate towards Phe and Tyr biosynthesis is generally signifi cantly stronger than the fl ux towards Trp biosyn-thesis, and the fl ux from arogenate towards Phe biosynthesis is signifi cantly stronger than that into Tyr biosynthesis (Rippert et al., 2004) (Fig. 7). This may also refl ect the fact that Phe pro-duces a signifi cantly larger variety of secondary metabolites than Tyr and Trp.

The enzymes of the shikimate and AAA biosynthesis pathways are generally synthesized as precursors containing a plastid tran-sit peptide that directs them to the plastid, the organelle in which these two essential pathways operate (Mustafa and Verpoorte, 2005; Weber et al., 2005; Zybailov et al., 2008). However, the intra-cellular localization of two enzymes, CM2 and ADT3, is still under some debate. Sub-cellular fractionation analysis suggested that the tobacco CM2 isozyme is localized in the cytosol (d’Amato et al., 1984), but whether this polypeptide indeed possesses CM

activity has not been confi rmed. Although in vitro studies showed that AtCM2 possesses CM activity (Eberhard et al., 1993; Eber-hard et al., 1996), the physiological signifi cance of AtCM2 still remains questionable (Rippert et al., 2009). In addition, an Ara-bidopsis polypeptide termed PDT1 (which corresponds to the ADT3 isozyme of Phe biosynthesis, characterized by Cho et al. 2007), was suggested to be a component of the heterotrimeric G-protein complex that is associated with the plasma membrane (Warpeha et al., 2006). This observation is in contrast to a more recent report, using an in situ microscopy analysis, which showed that all of the Arabidopsis ADT isozymes are localized in the plas-tid (Rippert et al., 2009). Thus, the current dogma is that all ADT isozymes are generally localized to the plastid, although it cannot be ruled out that under specifi c growth stages or physiological conditions, ADT3 may also be associated with other complexes before it is post-translationally transported into the plastid.

Infl uence of genetic, metabolic and environmental factors on the regulation of AAA metabolism

Several mutants and transgenic plants with modifi ed shikimate and AAA biosynthesis pathways were used to elucidate the regu-lation of the biosynthesis of the three AAA. A rice 5-methyl Trp-re-sistant mutant (Mtr1), apparently encoding a feedback-insensitive PDT/ADT, was shown to over-accumulate Phe and Trp in both callus tissue and leaves (Wakasa and Widholm, 1987; Yamada et al., 2008). Expression of a bacterial PheA* gene, encoding a bi-functional CM/PDT enzyme that is feedback insensitive to Phe, in transgenic Arabidopsis plants, caused: (i) signifi cant increases in the levels of Phe as well as a number of Phe-derived and Tyr-de-rived secondary metabolites; and (ii) signifi cant decreases of Trp-derived secondary metabolites (Tzin et al., 2009). This implied a regulatory cross-interaction between the biosynthesis fl uxes of the three AAA from chorismate, which also infl uence the rates of their conversion into various secondary metabolites. An Arabidop-sis double mutant lacking PAL1 and PAL2 activities has an ~100-fold increase in Phe and a 4-fold increase in Trp levels (Rohde et al., 2004). This pal1 and pal2 double mutant also infl uences the transcription of genes associated with the AAA biosynthesis network as well as genes associated phenylpropanoid secondary metabolites (Rohde et al., 2004). Arabidopsis and rice mutants with a feedback-insensitive ASa of Trp biosynthesis generally ac-cumulate Trp, but not Phe or Tyr (Kreps et al., 1996; Li and Last, 1996; Bender and Fink, 1998; Tozawa et al., 2001; Ishihara et al., 2006). Exposure of Arabidopsis seedlings to sulfate starvation triggers an increase in the level of shikimate as well as the Phe and Trp and secondary metabolites derived from them (Nikiforova et al., 2003; Nikiforova et al., 2004; Nikiforova et al., 2006).

CATABOLISM OF THE AROMATIC AMINO ACIDS INTO SEC-ONDARY METABOLITES

Phe catabolism

Phe serves as a precursor for a large family of secondary me-tabolites. The major group of these secondary metabolites is the phenylpropanoids, whose biosynthesis is initiated by the activ-



ity of Phe-ammonia lyase (PAL) (CE 4.3.1.5) (Fig. 8). Arabidop-sis possesses four genes encoding the PAL1-PAL4 isozymes (At2g37040, At3g53260, At5g04230 and At3g10340, respective-ly). The phenylpropanoids possess multiple functions, particularly protecting against various abiotic and biotic stresses, and their production is generally stimulated by such stresses (Dixon and Paiva, 1995; Dixon, 2001; Casati and Walbot, 2005). The tran-scription of the PAL genes is generally highly regulated by biotic and abiotic stresses, as well as by conditions that demand in-creased production of the cell wall component lignin in various tissues (Anterola and Lewis, 2002). Genetic mutations that affect the production of PAL generally cause signifi cant alteration in the levels of many phenylpropanoids (Shadle et al., 2003; Rohde et al., 2004). The major sub-groups of phenylpropanoids include the fl avonoids, the lignin cell wall components, and the antho-cyanins. The metabolite composition of the phenylpropanoids, as well as genes encoding enzymes and regulatory proteins as-sociated with their synthesis, have been recently discussed in several excellent reviews, examples of which are (Weisshaar and Jenkins, 1998; Pichersky and Gang, 2000; D’Auria and Gersh-enzon, 2005; Boudet, 2007; Vogt, 2010). Some decisive steps in phenylpropanoid biosynthesis were resolved only recently, such as the 2-hydroxylation involved in coumarate biosynthesis (Kai et al., 2008). In addition, genomics approaches revealed new or-gan-specifi c pathways, such as the formation of tapetum-specifi c trisacyl-polyamine conjugates of Arabidopsis fl ower buds (Alves-Ferreira et al., 2007; Ehlting et al., 2008; Fellenberg et al., 2009; Matsuno et al., 2009). The fi ne regulation of phenylpropanoid biosynthesis is achieved by combinatorial actions of transcription factors, expressed in a spatially and temporally controlled man-ner as exemplifi ed in the following reports: (Ramsay and Glover, 2005; Lepiniec et al., 2006; Stracke et al., 2007). A group of vola-tile compounds, including methylbenzoate, phenylethylacetate and isoeugenol, is also among the phenylpropanoids produced by PAL (Verdonk et al., 2003; Schuurink et al., 2006; Wildermuth, 2006; Ben Zvi et al., 2008).

Another class of sulfur-rich Phe-derived secondary metabo-lites includes the Phe-glucosinolates, whose basic skeleton consists of a b-thioglucose residue, an N-hydroxyiminosulfate moiety and a variable side chain (Reichelt et al., 2002). Phe-

glucosinolates are generally not widespread in Arabidopsis, but some Arabidopsis ecotypes do synthesize these compounds, such as phenylethylglucosinolate in the leaves (Mikkelsen et al., 2004) and benzoyloxyglucosinolates in seeds (Kliebenstein et al., 2007). The committing gene in the biosynthesis of Phe-glucosin-olates is the cytochrome P450, CYP79A2 (At5g05260), encod-ing an N-hydroxylase (CE 1.14.13) (Fig. 8) that converts Phe into phenylacetaldoxime, the precursor of benzylglucosinolate (Witt-stock and Halkier, 2000).

Some plant species also produce the volatile Phe-derived sec-ondary metabolite 2-phenylethanol (Facchini et al., 2000; Wata-nabe et al., 2002; Baldwin et al., 2004; Kaminaga et al., 2006; Tieman et al., 2006; Gonda et al., 2010). However, 2-phenyletha-nol is produced in fl owers and/or fruits of specifi c plants, such as petunia, rose and tomato, and so far this volatile has not been detected in Arabidopsis.

Tyr catabolism

Tyr serves as a precursor of several families of secondary metab-olites, including tocochromanols (vitamin E), plastoquinones, iso-quinoline alkaloids and non-protein amino acids, and it has also been speculated that Tyr may also lead to the production of some phenylpropanoids (Fig. 9). The tocochromanols, which include both tocopherols and tocotrienols, are essential antioxidants in the diets of human and farm animals (Schneider, 2005; Della-Penna and Pogson, 2006; Mene-Saffrane and Dellapenna, 2009). The fi rst committed enzyme of tocochromanols biosynthesis from Tyr is Tyr-aminotransferase (CE 2.6.1.5) (At5g53970) (Lopukhina et al., 2001), which produces p-hydroxyPPY (Fig. 9) (Norris et al., 1995; Garcia et al., 1999). It has been suggested that p-hydroxyP-PY can also be synthesized from prephenate via an alternative biosynthesis pathway (Fig. 5) (Rippert and Matringe, 2002b; Rip-pert et al., 2004). If such a pathway indeed naturally exists, then p-hydroxyPPY can also be used for tocochromanols biosynthesis, bypassing Tyr (Fig. 5).

The Tyr catabolism pathway also produces isoquinoline alka-loids, which represent a large, diverse group of natural products found in ~20% of all plant species (Facchini et al., 2004). In Arabi-

Figure 8. Phe catabolism. Only the fi rst enzymes involved in several secondary metabolism pathways derived from Phe are indicated. A putative pathway in Arabidopsis is marked with a dashed grey line. PAL, Phe-ammonia lyase; AADC, aromatic amino acid decarboxylase.



dopsis, Tyr is also catabolized into tyramine by Tyr/L–dopa decar-boxylase (TYDC) (EC 4.1.1.25), which is encoded by two genes (At2g20340, At4g28680). Tyramine is a precursor for benzyl-iso-quinoline alkaloids, as well as cell wall-bound hydroxycinnamic acid amides (Facchini et al., 2000). It has been suggested that tyramine in involved in the Arabidopsis defense response (Trez-zini et al., 1993).

Even though phenylpropanoids are classically synthesized from Phe, it has also been proven that in several plant species the second metabolite of the phenylpropanoid pathway, name-ly coumarate, can also be synthesized directly from Tyr by Tyr ammonia-lyase (TAL) (EC 4.3.1.) (Neish, 1961; Beaudoin-Eagan and Thorpe, 1985; Guerra et al., 1985; Rosler et al., 1997; Khan et al., 2003; MacDonald and D’Cunha, 2007). All four isoforms of Arabidopsis PAL, the fi rst enzyme of phenylpropanoid biosyn-thesis from Phe (Fig. 8), exhibit higher affi nity for Phe than for Tyr (Cochrane et al., 2004). However, a point mutation in the Arabi-dopsis gene encoding the PAL1 isoform resulted in a lower PAL

activity and a compensatory increase in TAL activity (Watts et al., 2006), supporting the potential use of TAL in the phenylpropanoid biosynthesis pathway.

Trp catabolism

Trp is catabolized into many indole-containing secondary me-tabolites, such as indole-3-acetic acid (IAA, auxin) (Ostin et al., 1998; Davies, 2004), indole glucosinolates (Halkier, 1999), phyto-alexins (Pedras et al., 2000), terpenoid indole alkaloids (De Luca and St Pierre, 2000; Facchini et al., 2004), and tryptamine de-rivatives (Facchini et al., 2000) (Fig. 10). Auxins are some of the key metabolites synthesized from Trp. However, the biosynthetic pathway(s) leading to IAA, the main auxin metabolite, are not well understood. Although there is good evidence that IAA is synthe-sized from Trp (Gibson et al., 1972; Wright et al., 1991; Tsurusaki et al., 1997), several different routes of IAA biosynthesis from Trp

Figure 10. Trp catabolism. Only the fi rst enzymes involved in several secondary metabolism pathways derived from Trp are indicated. The numbers within the Trp catabolism pathway indicate: 1) the indole-3-acetaldoxime (IAOx) pathway catalyzed by two cytochrome P450s (CYP79B2 and CYP79B3); 2) the tryptamine (YUCCA) pathway catalyzed by Trp decarboxylase (TDC); 3) the indole-3-pyruvate (IPyA) pathway catalyzed by Trp aminotransferase (TAA); and 4) the indoleacetamide (IAM) pathway which initiates directly from Trp via either IAOx or indole-3-acetonitrile (IAN).

Figure 9. Tyr catabolism. Only the fi rst enzymes involved in several secondary metabolism pathways derived from Tyr are indicated. Putative pathways in Arabidopsis are marked with dashed grey lines. TyrAT, Tyr-aminotransferase; TYDC, Tyr/L–dopa decarboxylase; PDH, prephenate dehydrogenase; TAL, Tyr-ammonia lyase.



Table 1. Arabidopsis thaliana genetic loci and enzyme activities mentioned in this review.

Pathway Step Gene name AGI locus ID CE Proven or predicted activities in A.thaliana

Shikimate 1 DAHPS1 At4g39980 2.5.1.54 3-Deoxy-D-arabino-heptulosonate-7-phosphate synthase Shikimate 1 DAHPS1 At1g39981 2.5.1.54 3-Deoxy-D-arabino-heptulosonate-7-phosphate synthase (putative) Shikimate 1 DAHPS2 At4g33510 2.5.1.54 3-Deoxy-D-arabino-heptulosonate-7-phosphate synthase Shikimate 2 DHQS At5g66120 4.2.3.4 3-Dehydroquinate Synthase Shikimate 3 DHQ/SDH At3g06350 4.2.1.10 3-Dehydroquinate dehydratase//Shikimate 5-dehydrogenase Shikimate 4 DHQ/SDH At3g06350 1.1.1.25 3-Dehydroquinate dehydratase//Shikimate 5-dehydrogenase Shikimate 5 SK1 At2g21940 2.7.1.71 Shikimate Kinase Shikimate 5 SK2 At4g39540 2.7.1.71 Shikimate Kinase Shikimate 6 EPSPS At2g45300 2.5.1.19 5-Enolpyruvylshikimate 3-phosphate Synthase Shikimate 6 EPSPS At1g48860 2.5.1.19 5-Enolpyruvylshikimate 3-phosphate Synthase (putative) Shikimate 7 CS At1g48850 4.2.3.5 Chorismate Synthase

Aromatic amino acids

Phe/Tyr 1 CM1 At3g29200 5.4.99.5 Chorismate Mutase Phe/Tyr 1 CM2 At5g10870 5.4.99.5 Chorismate Mutase Phe/Tyr 1 CM3 At1g69370 5.4.99.5 Chorismate Mutase

Phe option 1:

Phe - Arogenate route 2 PAT - 2.6.1.79 Prephenate Aminotransferase Phe - Arogenate route 3 ADT1 At1g11790 4.2.1.49 Arogenate Dehydratase Phe - Arogenate route 3 ADT2 At3g07630 4.2.1.49 Arogenate Dehydratase Phe - Arogenate route 3 ADT3 At2g27820 4.2.1.49 Arogenate Dehydratase Phe - Arogenate route 3 ADT4 At3g44720 4.2.1.49 Arogenate Dehydratase Phe - Arogenate route 3 ADT5 At5g22630 4.2.1.49 Arogenate Dehydratase Phe - Arogenate route 3 ADT6 At1g08250 4.2.1.49 Arogenate Dehydratase

Phe option 2:

Phe - Phenylpyruvate route 2 ADT1 At1g11790 4.2.1.49 Arogenate Dehydratase//Prephenate Dehydratase Phe - Phenylpyruvate route 2 ADT2 At3g07630 4.2.1.49 Arogenate Dehydratase//Prephenate Dehydratase Phe - Phenylpyruvate route 2 ADT6 At1g08250 4.2.1.49 Arogenate Dehydratase//Prephenate Dehydratase Phe - Phenylpyruvate route 3 AAAAT - 2.6.1.57 Aromatic Amino Acid Aminotransferase

Tyr option 1:

Tyr - Arogenate route 2 PAT - 2.6.1.79 Prephenate Aminotransferase Tyr - Arogenate route 3 TyrA1, ADS1 At5g34930 1.3.1.43 Arogenate Dehydrogenase Tyr - Arogenate route 3 TyrA2, ADS2 At1g15710 1.3.1.43 Arogenate Dehydrogenase

Tyr option 2:

Tyr - p-Hydroxyphenylpyruvate route 2 PDH - 1.3.1.43 Prephenate Dehydrogenase Tyr - p-Hydroxyphenylpyruvate route 3 AAAAT - 2.6.1.57 Aromatic Amino Acid Aminotransferase

Trp

Trp 1 ASa1, AMT1, TRP5 At5g05730 4.1.3.27 Anthranilate Synthase alpha subunit 1 Trp 1 ASa2 At2g29290 4.1.3.27 Anthranilate Synthase alpha subunit 2 Trp 1 ASa At2g28880 4.1.3.27 Anthranilate Synthase alpha subunit (putative) Trp 1 ASa At3g55870 4.1.3.27 Anthranilate Synthase alpha subunit (putative) Trp 1 ASb1, TRP4 At1g25220 4.1.3.27 Anthranilate Synthase beta subunit 1 Trp 1 ASb At1g25155 4.1.3.27 Anthranilate Synthase beta subunit (putative) Trp 1 ASb At1g25083 4.1.3.27 Anthranilate Synthase beta subunit (putative) Trp 1 ASb At1g24909 4.1.3.27 Anthranilate Synthase beta subunit (putative) Trp 1 ASb At1g24807 4.1.3.27 Anthranilate Synthase beta subunit (putative) Trp 1 ASb At5g57890 4.1.3.27 Anthranilate Synthase beta subunit (putative) Trp 2 PAT1, TRP1 At5g17990 2.4.2.18 Anthranilate Phosphoribosyltransferase Trp 3 PAI1 At1g07780 5.3.1.24 Phosphoribosylanthranilate Isomerase Trp 3 PAI2 At5g05590 5.3.1.24 Phosphoribosylanthranilate IsomeraseTrp 3 PAI3 At1g29410 5.3.1.24 Phosphoribosylanthranilate Isomerase Trp 4 IGPS At2g04400 4.1.1.48 Indole-3-Glycerol Phosphate Synthase Trp 4 IGPS At5g48220 4.1.1.48 Indole-3-Glycerol Phosphate Synthase (putative) Trp 5 TSa, TRP3 At3g54640 4.2.1.20 Tryptophan Synthase alpha subunit

(continued on next page)



have been proposed (Fig. 10) (Strader and Bartel, 2008; Quit-tenden et al., 2009). These include: 1) the indole-3-acetaldoxime (IAOx) pathway catalyzed by two cytochrome P450s (CE 1.14.13) (CYP79B2 and CYP79B3; At4g39950 and At2g22330) (Hull et al., 2000; Bartel et al., 2001); 2) the tryptamine (YUCCA) pathway catalyzed by Trp decarboxylase (TDC) (CE 4.1.1.28) (Facchini et al., 2000; Quittenden et al., 2009); 3) the indole-3-pyruvate (IPyA) pathway catalyzed by Trp aminotransferase (TAA)(CE 2.6.1.1) (At1g70560) (Stepanova et al., 2008; Tao et al., 2008); and 4) the indoleacetamide (IAM) pathway which initiates directly from Trp via either IAOx or indole-3-acetonitrile (IAN) (Pollmann et al., 2002). In addition, a possible additional, Trp-independent path-way of IAA biosynthesis directly from indole has been proposed (Normanly et al., 1993; Radwanski et al., 1996).

Another important group of secondary metabolites derived from Trp includes the glucosinolates, which are amino acid-de-rived natural plant products containing a thio-Glc moiety and a sulfonate moiety bound to an oxime function (Halkier and Gersh-enzon, 2006). They are implicated in plant-insect and plant-patho-gen interactions, and also recently attracted attention as cancer-preventive agents in humans (Halkier, 1999). Glucosinolates are found almost exclusively in the Brassicales and have been widely studied in Arabidopsis and in other species of the Brassicaceae family (Rask et al., 2000; Reichelt et al., 2002; Yatusevich et al., 2009). The IAOx, described above is also channeled by the ox-ime-metabolizing CYP83B1 enzyme into the biosynthetic path-way of indole glucosinolates (Naur et al., 2003).

The Trp catabolic pathway also synthesizes camalexin, the major indolic phytoalexin in Arabidopsis accumulating upon infec-tion with plant pathogens and abiotic elicitors (Zhao and Last,

1996; Bottcher et al., 2009). Camalexin originates from IAOx (Fig. 10) (Hull et al., 2000; Mikkelsen et al., 2000; Zhao et al., 2002). In addition, the Trp catabolic pathway also leads to the synthesis of indole alkaloids via tryptamine (Fig. 10). One example of the Trp-derived indole alkaloids is vindoline, an important metabolite in human health (Facchini et al., 2000; Facchini et al., 2004; Malitsky et al., 2008; Sugawara et al., 2009). However, indole alkaloids are generally not found in Arabidopsis.

FUTURE PROSPECTS

The entire set of genes and enzymes associated with the shiki-mate pathway have been elucidated (Table 1). However, elucida-tion of the regulation of this pathway is still in its infancy, requiring future studies. Even though, there were signifi cant discoveries as-sociated with genes and enzymes of the biosynthesis of the AAA in recent years, there are still missing links and debates about some key regulatory steps. The major route of Phe biosynthesis occurs through arogenate, but gene(s) encoding prephenate ami-notransferase have yet to be identifi ed. In addition, due to the fact that some plant arogenate dehydratase isozymes also possess residual prephenate dehydrate activities, as well as the observa-tion that plants apparently possess aminotransferase activity that can convert PPY into Phe, one cannot rule out a minor contribu-tion of a bacterial-like PPY route to Phe biosynthesis in plants. In addition, future studies should identify whether arogenate is the precursor for Tyr biosynthesis or whether an alternative bacterial-like route of Try biosynthesis via p-hyroxyPPY also exists. The regulation of the fl ux balance in the conversion of chorismate into

Table 1. (continued)

Pathway Step Gene name AGI locus ID CE Proven or predicted activities in A.thaliana

Trp 5 TSa, INS At4g02610 4.2.1.20 Tryptophan Synthase alpha subunit (putative) Trp 6 TSb1, TRP2 At5g54810 4.2.1.20 Tryptophan Synthase beta 1 subunit Trp 6 TSb2 At4g27070 4.2.1.20 Tryptophan Synthase beta 2 subunit Trp 6 TSb At5g28237 4.2.1.20 Tryptophan Synthase beta subunit (putative) Trp 6 TSb At5g38530 4.2.1.20 Tryptophan Synthase beta subunit (putative)

Phe catabolism (only representative enzyme)

Phenylpropanoids PAL1 At2g37040 4.3.1.5 Phe-Ammonia-Lyase1 Phenylpropanoids PAL2 At3g53260 4.3.1.5 Phe-Ammonia-Lyase2 Phenylpropanoids PAL3 At5g04230 4.3.1.5 Phe-Ammonia-Lyase3 Phenylpropanoids PAL4 At3g10340 4.3.1.5 Phe-Ammonia-Lyase4 Phe-glucosinolates CYP79A2 At5g05260 1.14.13.- cytochrome P450 CYP79A2

Tyr catabolism (only representative enzyme)

Tocochromanols TyrAT At5g53970 2.6.1.5 Tyr-Aminotransferase Benzyl-isoquinoline alkaloids TYDC At2g20340 4.1.1.25 Tyr/L–Dopa Decarboxylase Benzyl-isoquinoline alkaloids TYDC At4g28680 4.1.1.25 Tyr/L–Dopa Decarboxylase

Trp catabolism (only representative enzyme)

Auxin - indole-3-acetaldoxime (IAOx) CYP79B2 At4g39950 1.14.13.- cytochrome P450s CYP79B2pathway//Camalexin//Indole glucosinolate Auxin - indole-3-acetaldoxime (IAOx) CYP79B3 At2g22330 1.14.13.- cytochrome P450s CYP79B3pathway//Camalexin//Indole glucosinolate Auxin - indole-3-pyruvic acid (IPyA) pathway TAA At1g70560 2.6.1.1 Trp Aminotransferase



Trp and Phe/Tyr has already been extensively studied. Yet, the fl ux balance regulating the conversion of arogenate into either Phe or Tyr is still unknown, requiring future studies. Although rela-tively old studies suggest the presence of several enzyme feed-back inhibition loops within the AAA biosynthesis pathway, some studies provide clues for additional ones (Fig. 7), which require future confi rmation. Finally, a number of transcription factors have been proven to control different steps in the biosynthesis of AAA and secondary metabolites derived from them. Yet, it is likely that these do not represent the full set and additional studies are re-quired to address this issue. Interestingly, some transcription fac-tors regulate genes encoding both primary and secondary metab-olism associated with the AAA, and an exciting prospect for future research would to test whether the primary metabolism enzymes regulated by these transcription factors represent key regulatory enzymes connecting primary and secondary metabolism.

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